Thymidylate synthetase (TSase) is an enzyme ubiquitous to all cells which is essential for the synthesis of DNA. Information on the detailed thermochemistry of the transformation of deoxyuridylate (dUMP) to deoxythmidylate (dTMP) has recently been deduced in this laboratory. The proposed research will probe more deeply into this important reaction by specifically examining the binding of the substrates to the enzyme. An attempt will also be made to determine which of several oligoglutamylfolates, the in vivo cofactor, binds with the greatest affinity. The proposed research will also examine the quaternary structure of the enzyme and monitor the effect of chemical modification on both the quaternary structure and the catalytic activity of the enzyme. This latter information will be of value in furthering our understanding of uncontrolled cellular replication. The physical chemistry of proteins has evolved to a point such that questions concerning the relation of structure and function may be asked and in many cases answers found. Thymidylate synthetase is an ideal model for asking thermodynamic questions concerning structure/function relations. The enzyme contains two subunits and is thus a simple model for studying the equilibrium: iAl reversible yields Ai; (i equals 2). Furthermore, by the novel combination of ultracentrifugation and microcalorimetry, all state functions, delta G degrees , delta H degrees, delta S degrees, and delta C degrees p for this reaction may be deduced. A combination of the above thermodynamic measurements with specific chemical modifications of the enzyme will lead to detailed information relating specific amino acids residues with the quaternary structure of TSase. Recent advances in theoretical descriptions of quaternary structure require precise determinations of delta G degrees and delta H degrees for critical evaluation. The proposed research will supply these data. Currently, very little information exists concerning the enthalpy changes accompanying both substrate binding and catalysis for any single reaction. The several, varied chemical steps in the reaction dUMP plus CH2THF yields sub TSase dTMP plus DHF (1) make this an exciting reaction for calorimetric study. Furthermore, the metabolic significance of thymidine synthesis and its relevance to DNA biosynthesis stimulates our interest in these basic thermodynamic measurements.